The Ttg2 ABC system in Pseudomonas aeruginosa functions as a general glycerophospholipid transporter between the two cell membranes

In Pseudomonas aeruginosa, Ttg2D is the soluble periplasmic phospholipid-binding component of an ABC transport system thought to be involved in maintaining the asymmetry of the outer membrane. The crystallographic structure of Ttg2D at 2.5 Å resolution reveals that this protein can accommodate four acyl chains. Analysis of the available structures of Ttg2D orthologs shows that they conform a new substrate-binding-protein structural cluster. Native and denaturing mass spectrometry experiments conrm that Ttg2D, produced both heterologously and homologously and isolated from the periplasm, can carry two diacyl glycerophospholipids as well as one cardiolipin. Binding is notably promiscuous, allowing the transport of various molecular species. In vitro binding assays coupled to native mass spectrometry show that binding of cardiolipin is spontaneous. Gene knockout experiments in P. aeruginosa multidrug-resistant strains reveal that the Ttg2 system is involved in low-level intrinsic resistance against certain antibiotics that use a lipid-mediated pathway to permeate through membranes.


Introduction
Pseudomonas aeruginosa are amongst the most important multidrug-resistant (MDR) human pathogens 1 , showing inherent resistance to an important fraction of the available antibiotics 2 . P. aeruginosa are responsible for chronic lung infections in individuals with chronic obstructive pulmonary disease or cystic brosis (CF) 3 and account for over a tenth of all nosocomial infections 4 . A number of effective drugs and formulations can treat P. aeruginosa infections, even in CF patients 5 . These include frontline antibiotics such as piperazillin-tazobactam, ceftazidime, aztreonam, imipenem, meropenem, cipro oxacin, levo oxacin, tobramycin, amikacin, and colistin 6 . Yet, resistance to most of these antimicrobials is being increasingly reported 7 . The basis for the inherently high resistance of these microorganisms is primarily their low outer-membrane (OM) permeability 8,9 , complemented by the production of antibiotic-inactivating enzymes (e.g. β-lactamases), the constitutive expression of e ux pumps 10,11 and the capacity to form bio lms 1,12 , among other mechanisms. The susceptibility of P. aeruginosa to antimicrobials can be additionally reduced by the acquisition of inheritable traits, including horizontal gene transfers and mutations that decrease uptake and promote e ux pump overexpression 13,14,15 . Although a number of genes and mechanisms of resistance to antibiotics are already known in P.
aeruginosa, the complex mechanisms controlling the basal, low-level resistance to these compounds are still poorly understood 16,17 .
The OM of P. aeruginosa is known to be central to its antibiotic-resistance phenotype. Its intrinsically low permeability is partly determined by ine cient OM porin proteins that provide innate resistance to several antimicrobial compounds, mainly of hydrophilic nature 1,8,10 . In addition, studies with mutant strains have shown that the loss of speci c e ux pump mechanisms, commonly overproduced in clinical isolates, is compensated by reducing the permeability of the OM 9 . Furthermore, polymyxin resistance in P. aeruginosa is associated with signi cant alteration of the membrane's glycerophospholipid composition 18 . Thus, mechanisms involved in OM organization, composition and integrity interfere with the diffusion through the membrane of both hydrophilic and hydrophobic antimicrobial compounds. The asymmetry in the lipid organization of the OM is a main factor in the low permeability to lipophilic antibiotics and detergents 19 . Glycerophospholipid tra cking across bacterial membranes contributes to that asymmetry, but the systems involved in this transport and their directionality are just beginning to be understood.
In Escherichia coli, the Mla system (MlaA-MlaBCDEF) was initially proposed to have a phospholipid import function, preventing phospholipid accumulation in the outer lea et of the OM and thus controlling membrane-phospholipid asymmetry 20 . The core components of this ATP-binding-cassette (ABC) transport system in the inner membrane (IM) comprise the permease (MlaE), the ATPase (MlaF) and the substrate-binding protein MlaD that are highly conserved among Gram-negative bacteria 21 . The MlaA component, an integral OM protein that forms a channel adjacent to trimeric porins, would selectively remove phospholipids from the outer OM lea et and transfer them to the soluble periplasmic substratebinding protein MlaC 22,23 . MlaC would then transport the phospholipids across the periplasm and deliver them to MlaD for active internalization through the IM 24 . Deletion of the genes of this system is known to destabilize the OM, and bacterial strains lacking any of the Mla components are more susceptible to membrane stress agents 20,25,26,27,28,29,30,31 . More recently, the retrograde transport hypothesis has been questioned and a new role for this system in anterograde phospholipid transport has been suggested. Thus, the Mla system in E. coli and its homolog in Acinetobacter baumannii appear to participate in the export of phospholipids to the OM 30,32 . In this context, MlaD would extract phospholipids from the IM and transfer them to MlaC in an ATP-independent manner. The orthologous Mla system in P. aeruginosa is encoded by the PA4452-PA4456 operon (locus tags corresponding to PAO1) and the isolated gene PA2800 (MlaA ortholog, also known as VacJ). Proteins encoded by this gene cluster are highly similar to those encoded by operon ttg2 (toluene tolerance genes) in Pseudomonas putida 33,34 . Although it is unlikely that organic solvents themselves are substrates of this transporter, this system was initially linked to toluene tolerance in this species 35 . Accordingly, components of the P. aeruginosa ABC transporter encoded by the PA4452-PA4456 have been named Ttg2A (MlaF), Ttg2B (MlaE), Ttg2C (MlaD), Ttg2D (MlaC) and Ttg2E (MlaB) 33 . Recent studies of mutant strains with disrupted ttg2 or vacJ genes support the contribution of this ABC transport system to the intrinsic resistance of P. aeruginosa to antimicrobials 25,29,33,36 . Nevertheless, one of these studies has challenged the role of this system in intermembrane phospholipid tra cking in P. aeruginosa 33 .
Here, we have primarily focused on P. aeruginosa's Ttg2D (Ttg2D Pae ) and the nature of its cargo, which shall provide additional clues on the function of the Ttg2 system as a whole. Thus, we present structural and functional evidence of the role of this protein as a general phospholipid transporter, capable of carrying either diacyl or tetra-acyl glycerophospholipids. Our structural analysis further enriches the existing knowledge on the structural diversity of substrate-binding proteins (SBPs) and supports current discussions on the directionality of phospholipid transport by the Mla system. In addition, based on mutational studies of the ttg2 operon, we have validated the contribution of the Ttg2 system to the intrinsic basal resistance of P. aeruginosa to several antibiotic classes and other damaging compounds. Although the role of other components of this ABC transport system in multi-drug resistance has been already established for P. aeruginosa 25,33 , this is the rst study focusing on the soluble periplasmic SBP component, Ttg2D Pae . Among the components of the Ttg2 system, this SPB could be the most promising candidate for an antimicrobial intervention based on the speci c blocking of this tra cking pathway.

Results
Ttg2D Pae contains a large hydrophobic cavity that binds four acyl tails Sequence analysis indicates that Ttg2D Pae (PA4453) is the soluble periplasmic SBP component of the ABC transporter encoded by the ttg2 operon and a member of the Pfam family MlaC (PF05494). Interestingly, the available 3D structures for the MlaC family from Ralstonia solanacearum (PDB entry 2QGU), P. putida (PDB entries 4FCZ and 5UWB) and E. coli (PDB entry 5UWA) were all solved in complex with a ligand in their hydrophobic pocket, except for one structure from E. coli (PDB entry 6GKI) where the protein was delipidated. Electron densities for the ligand were in all these structures compatible with phospholipid moieties, supporting their predicted role as phospholipid transporters. A sequence alignment shows that some of the residues thought to be involved in phospholipid binding in the R. solanacearum Ttg2D structure are conserved in the P. aeruginosa ortholog (Fig. S1). Remarkably, the electron densities for P. putida Ttg2D revealed the presence of two diacyl lipids in its pocket 27 .
To investigate ligand binding at the molecular level, we determined by molecular replacement the crystallographic structure of the Ttg2D Pae mature protein (without the signal peptide, aa  produced in E. coli at 2.53 Å resolution (PDB entry code 6HSY) (Fig. 1A). The structure was re ned to a nal R work and R free of 20.9 and 24.9%, respectively, and good validation scores (Supplementary Table   S1). All residues but the last three C-terminal ones (plus the C-terminal expression tag) could be modeled.
Ttg2D Pae adopts a mixed α + β fold with a highly twisted anti-parallel β-sheet formed by ve strands and surrounded by eight α-helices. It exhibits a "decanter" shaped structure never described before for any other protein family (Fig. 1A). The structure presents a highly hydrophobic cavity between the β-sheet and the helices that spans the whole protein and has a volume of 2979 Å 3 and a depth of ~ 25 Å (Fig. 1B).
After the rst re nement stage (AutoBuild), without any ligand added, clear density was visible inside the cavity that could correspond to four acyl chains (Fig. S2). We therefore modeled inside the cavity two PG(16:0/cy17:0) (Fig. 1A), as mass spectrometry (MS) experiments suggested that this lipid was one of the most abundant among those found to bind Ttg2D Pae when expressed in E. coli (see below). Realspace correlation coe cients of 0.9 for the lipids indicate a good t to the 2mF o -DF c electron density.
The four acyl tails are deeply inserted into the hydrophobic cavity, while the polar head groups are exposed to the solvent and make only few contacts with the protein (Fig. 1, A and C). This lack of speci c recognition of the head group could explain why Ttg2D Pae is able to bind different types of phospholipids. The presence of two diacyl lipids suggests that the protein could also be able to bind one tetra-acyl lipid, such as diphosphatidylglycerol (cardiolipin).
To investigate the mechanism of entry and release of the two lipids in the cavity of Ttg2D Pae , we performed a normal mode analysis (NMA). NMA may be used to model the internal collective motions of a protein, relevant to ligand biding and function in general, typically described by a few low-frequency modes 37 . Figure 1D shows the collective motions along mode 7, the rst non-trivial mode (modes 1 to 6 account for translational and rotational motions of the protein as a whole). Rather than "en bloc" relative motions of sub-domains, all secondary structures of the protein appear to move in a concerted manner, helix α4 and the core of the β-sheet being more rigid. This breathing-like motion increases in a concerted manner the volume of the cavity and its mouth area, and may allow the lipids to enter into or exit from the cavity. Inspection of the next 10 lowest-frequency normal modes shows similar concerted motions. The recent MalC structure with no lipid bound (PDB entry 6GKI) 32 shows similar collective motions along all modes, with similar amplitudes, indicating that the cavity could open and close in the absence of lipid. The normal modes can be also used to compute atomic mean-square displacements, which can be in turn related to B-factors 38 . The NMA-derived and the observed (crystallographic) B-factors are closely correlated except in regions 75-95 and 180-200, which are involved in crystal contacts, and region 105-120, where the electron density is weaker (Fig. S3). This suggests that the normal modes provide a realistic description of the protein's exibility.
The Ttg2D/MlaC fold: a new two-domain architecture and SBP structural cluster All MlaC homologs of known structure have a highly superposable "decanter" shaped con guration ( Fig. 1A), previously described as an "extended" NTF2 fold 27 Fig. S1) of Ttg2D Pae against the whole PDB. The results showed very good superposition with the small alpha domain of several AAA + proteins, the best match being with 6UKS chain C 39 (Fig. S4). AAA (ATPases Associated with diverse cellular Activities) domains are formed by a large N-terminal domain adopting a Rossmann fold and a small C-terminal domain forming an alpha-helical bundle. They tend to adopt homo-hexameric ring complexes and hydrolyze ATP to perform activities that involve protein remodeling. The helical domain plays an important role in coupling the conformational changes resulting from ATP hydrolysis to the neighbor monomer within the AAA-ring and to the underlying protease ring 40 and has a speci c classi cation (other than AAA) both in the PFAM (PF17862) and CATH databases (1.10.8.60).
Interestingly, the Ttg2C ortholog MlaD, which in E.coli interacts with MlaC 32 , forms also a homo-hexameric ring. We therefore conclude that currently known Ttg2D/MlaC structures combine two structural domains in an architecture not seen yet in other proteins, a NTF2-like domain and an AAA helical-bundle domain. In Ttg2D Pae , the rst domain is formed by two non-contiguous sequence segments: D1S1 (PDB residues 23-68), with three alpha-helices, and D1S2 (113-169), with ve betastrands. The second domain, with ve helices, is also split in two non-contiguous sequence segments: D2S1 (69-112) and D2S2 (170-212) ( Fig. 1A and S1).
Classical SBPs linked to ABC transporters are structurally similar and composed of two globular domains formed by discontinuous segments 41  Evolution of sequence and structural diversity of the MlaC family A structural alignment of MlaC family proteins with known 3D structures ( Fig. 2A) reveals that, despite sequence identities ranging from 63% for the P. putida protein to as low as 17% for the E. coli one, the RMSDs of the structural alignments are very low, ranging from 1.6 to 3.1 Å (188 to 185 Cα), respectively (Table S2). Clearly, secondary structure elements are highly or strictly conserved among all four proteins, despite substantial amino-acid variation ( Fig. 2A and S1). However, the four proteins split into two groups: P. aeruginosa and P. putida Ttg2D have a hydrophobic cavity of 2979 − 2337 Å 3 and can bind two diacyl lipids, while the R. solanacearum and E. coli proteins have a half-size cavity of 1444 − 1332 Å 3 and bind only one diacyl lipid (Table S2). Surprisingly, although the different number of ligands had been already noticed when the structure of Ttg2D from P. putida was solved, cavity differences were never analyzed. Figure 1B illustrates the cavity difference between P. aeruginosa and R. solanacearum Ttg2D. The volume differences correlate with the different number of residues forming the cavities, from 55 down to 31 (Table S2). However, these residues, which are spread along the whole protein sequence (Fig.  S1), are largely conserved in terms of position and, in most cases, in terms of identity or similarity, with a few substitutions such as V147/L, or V163/I or M directly affecting the volume. Some side-chain reorientations, like Y105, and small secondary structure displacements, like strands β3 and β4 or helix α6 shifted by ~ 2 Å ( Fig. 2A and S6), also modulate the volume. Taken together these changes are, nevertheless, not su cient to explain how the cavity volume can double. Helix α8 seems to be the main responsible for the difference between a two and a one diacyl-phospholipid cavity, not only because the helix is longer in the rst case ( Fig. S1), but also because it adopts a different conformation. Indeed, for the second group (R. solanacearum and E. coli, one diacyl lipid), this helix has a straight conformation, covers the α6 helix ( Fig. 2A and S6) and does not participate in the cavity ( Fig. 1B and S1), while in the rst group (P. aeruginosa and P. putida, two diacyl lipids), the α8 helix is bent towards and over the α7 helix and greatly enlarges the cavity (with additional residues from β4 and β5 strands). This bend occurs at residue G195 with an angle of 40º and 64º in Ttg2D proteins from P. aeruginosa and P. putida, respectively ( Fig. 2A). The helix of the rst protein has an additional bend of 43º at K202. Glycine has a poor helix-forming propensity 43 and tends to disrupt helices because of its high conformational exibility. On the other hand, the Phe197 and Gln196 residues occupying the Gly195 position in the R. solanacearum and E. coli proteins, respectively (Fig. S1), have better helix-forming propensities and maintain the α8 helix straight. In addition, W196, exclusive of the pseudomonal structures, may also contribute to the in uence of the α8 helix on the cavity's volume, since its bulky hydrophobic side chain, deeply inserted into a hydrophobic pocket on the concave side of the curvature, could stabilize the helix α8 bend (Fig. S6).
Components of the Mla system are broadly conserved in Gram-negative bacteria, except for the periplasmic MlaC that notoriously shows high inter-species sequence diversity (Fig. 2B). Interestingly, an alignment of 151 representative amino-acid sequences belonging to the MlaC family and identi ed across different Gram-negative species revealed that W196 is conserved not only in Pseudomonas species but also in a group of related sequences in other non-phylogenetically related gammaproteobacteria (Fig. 2C). In this group of proteins that would hypothetically bind two diacyl phospholipids, other positions with distinct residues relative to other MlaC family members stand out, especially in two regions located between the central part and the C-terminal end of the protein (Fig. S6). Side-chain orientation and hydrophobicity of some residues in these regions could be also contributing to a tighter binding of the two diacyl phospholipids inside the ligand cavity. The presence of common protein sequence signatures in species that are not closely related indicates that horizontal gene transfer, mediated by recombination events between anking conserved genes, could have contributed to MlaC family diversity.
Ttg2D Pae binds two diacyl glycerophospholipids and cardiolipin, representing a novel phospholipid tra cking mechanism among Gram-negative bacteria Native MS was used to determine the lipids that bind speci cally to recombinant Ttg2D Pae produced in the cytoplasm of E. coli and the stoichiometry of the interaction in a cellular environment (Fig. 3). The native mass spectrum of Ttg2D Pae shows two major charge states (m/z 2701, z = 9 and m/z 2430, z = 10) corresponding to a deconvoluted average mass of 24296 Da that matches the MW of the recombinant protein Ttg2D Pae produced in E. coli without the rst methionine residue plus bound ligands ( Fig. 3A and S7). After isolation of the wide peak ion at m/z 2700 (z = 9) and gas-phase fragmentation with a transfer collision energy (CE) of 50 V, we detected the unbound protein (m/z 2536, z = 9) and a family of released phospholipids in the low mass range (Fig. 3B-C). Additional native MS experiments on isolated ion m/z 2700 (z = 9) using increasing transfer CE con rmed that at least a fraction of the bound Ttg2D Pae population hosts two phospholipids, since lipid dissociation starts at a transfer CE of 35 V and goes through the single-bound species (Fig. S8). The results also show that a second fraction of the Ttg2D population hosts a single molecule, that remains bound up to a transfer CE of 50 V, which could correspond to cardiolipin as further MS experiments indicated. A tighter binding of cardiolipins could explain why these molecules were not detected in the gas-phase fragmentation experiments ( Fig. 3B-C). We also note that at the transfer CE required for cardiolipin release these molecules are easily fragmented (see below).
The mass of [M + H] + ions at m/z 664.5, 704.5, 718.5 and 730.5 released from Ttg2D Pae con rmed the identity of some ligands as phosphatidylethanolamines (PE) with different hydrocarbon chains (PE C30:0, PE C33:1, PE C34:1 and PE C35:2 respectively) (Fig. 3C). Subsequent direct MS analysis under denaturing conditions in both positive and negative ion modes, identi ed additional species of PE and of other phospholipid classes that are also components of the bacterial membrane, such as phosphatidylglycerol (PG), phosphatidylcholine (PC) and phosphatidylserine (PS) (Fig. 3D and S9). With these methods, PG C33:1, PG C34:1, PE C33:1 and PE C35:1 came out as most abundant (annotation of lipid species based on the most abundant fatty acids in E. coli is given in Table S3). The observed distribution of phospholipids bound to recombinant Ttg2D Pae relates to their relative abundances in E.
coli BL21 (the recombinant protein-expression host) as determined by lipidome analyses (Fig. S10), and it correlates well with the reported phospholipid composition of E. coli under comparable conditions 44,45 .
LC-MS analysis under denaturing conditions in positive ion mode of the complexes produced in E. coli shows lipid species of m/z 690-800 and m/z 1400-1500 that could correspond to phospholipids and cardiolipins, respectively (Fig. S9D). In addition, direct MS analysis in negative ion mode indicates the presence of glycerophospholipids and shows peaks at m/z 1050-1160 that could correspond to E. coli cardiolipin species having lost one fatty acid (Fig. 3D-E). During the fragmentation processes of phospholipids in negative ion mode, upon low energy collisional activation, ions corresponding to the loss of fatty acids are the most abundant 46 . For example, when the most abundant cardiolipin species in E. coli, i.e. CL C68:2 with MW 1405.0 Da (Fig. S10), loses one of the fatty acids at position sn-2, it shows a prominent fragment ion at m/z 1147 or 1121 depending on the fatty acid species in that position ( Fig. 3D-E).
In light of these results, we performed additional native MS experiments to investigate whether delipidated Ttg2D Pae can bind cardiolipin in vitro. In Fig. 4, representative native mass spectra of a reaction mixture containing 26.25 µM delipidated protein and 245 µM cardiolipin CL(18:0) 4 (1:9 molar ratio) in a buffered aqueous solution, are shown. The protein-cardiolipin complex and the unbound protein were detected around the 10 + and 9 + charge states (Figs. 4 and S11). Notably, the dissociation energy needed to release cardiolipin was higher than that required to release diacyl glycerophospholipids in the native complex (transfer CE of 60 V vs 35 V, see Figs. S11A and S8, respectively). This correlates with the previous observation that a "P + 2PL" population in the native spectra in Fig. S8 loss its cargo only at 60 V. At the transfer CE required for cardiolipin release this molecule is in fact easily fragmented (Fig. S11B).
Ttg2D Pae binds two diacyl glycerophospholipids or cardiolipin in the periplasm of P. aeruginosa Given that the cytoplasm of E. coli is clearly not the natural environment of Ttg2D Pae , we decided to produce and purify this protein directly from the periplasmic space of P. aeruginosa. For this purpose, protein Ttg2D Pae tagged with six C-terminal histidine residues and containing its own N-terminal secretion signal was expressed in the genetic background of a P. aeruginosa PAO1 Δttg2D mutant using a new derivative of a broad-host-range cloning vector (Fig. 5A). Restoration of the colistin susceptibility phenotype after transformation (MIC of 0.25 µg/ml vs. 0.0625 µg/ml in the non-transformed mutant) was used as evidence that the protein being expressed in the mutant strain was functional. Under these conditions, the mature Ttg2D Pae could be puri ed from the periplasm in su cient amount and purity ( Fig. 5B inset) to determine its phospholipid cargo by MS.
The native mass spectrum in Fig. 5B shows a charge-state distribution that corresponds to the ligandbound mature Ttg2D Pae complexes (MW of intact complex: 23621 Da; MW of His-tagged protein without the signal peptide: 22140 Da). The wide peak widths, besides poor desolvatation in aqueous buffer in the native MS instrumental conditions, suggests also the coexistence of multiple species of similar mass, supporting the presence of different classes of phospholipids bound to the protein. Gas phase dissociation mass spectra at varying transfer CE of isolated ions around m/z = 2625 (z = 9) indicated the presence of two populations at peaks m/z = 2626 (z = 9) and m/z = 2619 (z = 9), which are in agreement with the binding of two phospholipids and one cardiolipin, respectively (Fig. 5C). Further MS experiments under denaturing conditions in negative ion mode allowed the identi cation of two main phospholipid classes ( Fig. S12 and Table S4). The obtained distribution is in agreement with relative abundances in P. aeruginosa PAO1 as determined by a lipidome analysis (Fig. S10), and it correlates well with the reported phospholipid composition in this species 47 .
The Ttg2 system provides P. aeruginosa with a mechanism of resistance to membrane-damaging agents As expected, the P. aeruginosa Δttg2D mutant exhibited a debilitated outer membrane leading to increased susceptibility to several membrane damaging agents (Fig. 6), as demonstrated by the 1-Nphenylnaphthylamine (NPN) assay. Indeed, an enhancement in NPN uptake was observed in the mutant in the presence of the permeabilizer agents EDTA and colistin (Fig. 6, A and B). In line with this, the Δttg2D mutant is signi cantly more susceptible to the action of polymyxins (lipid-mediated uptake), but also of antibiotics that use both the lipid-and porin-mediated pathways to penetrate the cell, including uoroquinolones, tetracyclines and chloramphenicol (Fig. 6C). With regard to polymyxin antibiotics, the ttg2D transposon insertion mutant was eight-fold more susceptible to colistin than the PAO1 wild-type, a colistin-susceptible reference strain (Table S5). In general, the mutation did not signi cantly affect the resistance phenotype displayed by the PAO1 strain to the beta-lactam antibiotics or aminoglycosides tested. The susceptibility phenotypes due to deletion of ttg2D could be fully or partially reverted by complementation with the cloned ttg2D gene or the full operon ttg2 in the replicative broad-range vector pBBR1MCS-5 ( Fig. 6 and Table S5), con rming the link between the gene and the phenotypes. We have also con rmed that insertional mutations in each of the other components of the ttg2 operon (ttg2A, ttg2B, ttg2C) and vacJ (mlaA ortholog) lead to an increased susceptibility to antibiotics in the same way as for the Δttg2D mutant (Table S5). The Δttg2D mutant is also signi cantly susceptible to the toxic effect of the organic solvent xylene (Fig. 6D) and it is four-fold more susceptible to the chelating agent EDTA (MIC = 0.5 mM) than the parental wild-type PAO1. However, no difference was observed between the mutant and wild-type cells in their susceptibility to SDS, obtaining for both strains a MIC value of 0.8%. Finally, disruption of the ttg2D gene resulted in an approximately two-fold reduction in bio lm formation and greatly increased the activity of EDTA against P. aeruginosa bio lms at a subinhibitory concentration of 0.05 mM (Fig. 6E).
The Ttg2 system is associated to P. aeruginosa's intrinsic resistance to low antibiotic concentrations The susceptibility of Ttg2-defective mutants to antibiotics was further studied in strains with different genetic backgrounds. To this end, the full ttg2 operon was mutated in the clinical MDR P. aeruginosa strains C17, PAER-10821 and LESB58, which had shown different patterns of resistance to several antibiotic classes, speci cally, polymyxins, uoroquinolones and tetracyclines (Table 1). In particular, PAER-10821 and LESB58 are P. aeruginosa strains with low-level resistance to colistin. The generation of mutants with disrupted gene functions in MDR bacteria is troublesome because the antibiotics commonly used in the laboratory are no longer useful for selection of gene knockouts. In addition, the loci mutated in this case is involved in a general mechanism of resistance to antimicrobial agents and mutant strains are therefore expected to be generally susceptible and thus potentially lost during the selection steps. For this reason we have adapted a mutagenesis system based on the homing endonuclease I-SceI 48, 49 to construct targeted, non-polar, unmarked gene deletions in MDR P. aeruginosa strains (see material and methods, text S1 and Fig. S13 for details). With this modi ed mutagenesis strategy we have obtained and validated unmarked deletion mutants of the selected MDR strains lacking the full ttg2 operon (Fig. S13). Complemented strains were also obtained by transformation of mutant strains with a replicative plasmid containing the full ttg2 operon and its expression in the complemented clones was con rmed by RT-PCR (Fig. S13). All these strains were tested for their susceptibilities to different classes of antibiotics (Table 1). Ticarcillin-clavulanic acid > 32 > 32 > 32 > 32 > 32 > 32 † Minimum inhibitory concentration (MIC) determined by the broth microdilution method. MICs were con rmed by two or three independent replicates. MIC differences greater than 2-fold with respect to the corresponding wild type strain were considered signi cant (indicated with an asterisk).
The three ttg2 mutants were signi cantly more sensitive (between 4-and 64-fold) than the corresponding wild-type bacteria to colistin, uoroquinolones, and tetracycline analogues, but not to the other antibiotic classes ( Table 1). The mutant susceptibility phenotypes could be reverted by providing an intact copy of the entire PAO1 ttg2 operon (PA4456-PA4452) in a replicative plasmid, except for colistin. The lack of complementation of the colistin susceptibility phenotype could be due to the effect of the antibiotic erythromycin (used as a selection marker for complemented strains) on the expression of global regulators that may in uence colistin susceptibility 50,51 or to the overexpression of the ttg2 operon components (two-to eight-fold with respect to wild type, see Fig. S13) that may also affect the distribution of phospholipids in the OM. Surprisingly, the susceptibility to amikacin signi cantly decreased for the C17 mutant and an opposite effect was observed for the LESB58 mutant and tobramycin, suggesting a genetic-background component in the effect of the ttg2 mutation on the susceptibility to these antibiotics.

Discussion
We have performed a structural and functional study of the soluble periplasmic SBP component of the Ttg2 ABC transport system in P. aeruginosa (Ttg2D Pae ) that reveals new facets of this protein family and provides additional insight into the role of this pathway in P. aeruginosa. We have rst characterized this protein at the molecular level, supporting its predicted role as a phospholipid transporter. The crystal structure of recombinant Ttg2D Pae (Fig. 1), and MS analysis of protein-ligand complexes formed in vivo and in vitro (Fig. 3-5), show that this SBP transports four acyl chains. Furthermore, our results demonstrate that the Ttg2 system in P. aeruginosa is a general glycerophospholipid transporter with the ability to carry either two phospholipids or a tetra-acyl cardiolipin-like species. Reevaluation of the Ttg2D structure from P. putida (PDB 4FCZ) by Ekiert et al. 27 (PDB 5UWB) had previously suggested but not con rmed the presence of a tetra-acyl, cardiolipin-like lipid in its bulky hydrophobic pocket. This is in contrast with the Ttg2D/MlaC orthologs from E. coli (PDB 5WA) and R. solanacearum (PDB 2QGU), which seem to bind a single diacylglyceride based on electron density and cavity size. Although, more recently, Hughes et al. showed evidences of cardiolipin binding to E. coli MlaC 32 , these have yet to be con rmed, since the methodology used does not allow the distinction between speci c binding and co-puri cation (unspeci c). With the data at hand, it thus appears that, among Gram-negative bacteria, the ability of MlaC-family proteins to transport two phospholipids or cardiolipin is exclusive to some taxonomic groups. Phylogenetic and sequence analysis (Fig. 2) suggest that there are other genera in addition to Pseudomonas where the Mla system transports two molecules simultaneously, although this needs to be con rmed by further studies. This nding raises the question whether the evolution of this system in these species has been driven by transport e ciency (double cargo) or transport diversity (tetra-acyl in addition to diacyl phospholipids). Furthermore, are the two phospholipids translocated simultaneously by the permease Ttg2B, as it would need to be for a tetra-acyl phospholipid such as cardiolipin? The determination of the structure of additional transport components in other species will be necessary to corroborate our proposed classi cation and answer these questions.
MS analyses showed that Ttg2D Pae is a highly promiscuous SBP. This protein is indeed able to bind in vivo phospholipids with different head groups, particularly PG and PE, and of different chain lengths and degree of unsaturation. Therefore, this system would not only control the global phospholipid content of the OM, but could be also controlling a precise membrane-lipid distribution. Bacterial cells tightly regulate the phospholipid composition of the OM to fortify the permeability barrier against small toxic molecules, including antibiotics. For example, anionic phospholipids like PG interact with membrane proteins and cationic antimicrobials in ways that zwitterionic phospholipids like PE do not; their balance requiring a ne control 19,52 . Indeed, the membrane's PE content is a major factor determining the bacterial susceptibility to certain antimicrobial agents 52,53 . On the other hand, anionic non-bilayer-forming phospholipids like cardiolipin can strengthen the OM of Gram-negative bacteria against certain antibiotics 19 . In Gram-negative bacteria, cardiolipin is mostly located within the IM (the site of its synthesis), but it is also present in the OM where it facilitates proper localization of proteins on the bacterial surface. Known mechanisms of anterograde transport ensure the presence of cardiolipin in the OM 54 , but a retrograde mechanism preventing accumulation in the OM has not been described yet. An increase in the cardiolipin content of the OM could cause an increase in susceptibility to cationic antimicrobial agents 55 . Positively charged antimicrobial peptides and polymyxins have been proposed to promote the clustering of anionic phospholipids leading to phase-boundary defects that transiently breach the permeability barrier of the cell membrane 52 . In P. aeruginosa, an organism showing signi cant intrinsic resistance to certain antibiotics, the membrane PE:PG:cardiolipin composition is approximately 60%:20%:11% 18 . Simultaneous transport of four acyl chains across cell membranes could help control membrane charge balance more e ciently, not only by number but also by inclusion of additional (cardiolipin) species.
Early studies 20 (and more recent ones 56,57 ) of the Ttg2/Mla system supported its role in the maintenance of lipid asymmetry in the Gram-negative OM, by retrograde tra cking of phospholipids from the OM to the cytoplasm through the IM. Conversely, other studies have recently shown that the E. coli protein MlaD spontaneously transfers phospholipids to MlaC in vitro 32,56 , suggesting a transport in the opposite direction (IM-to-OM). In addition, an Mla mutant in A. baumannii showed decreased abundance of OM phospholipids and accumulation of newly synthesized phospholipids in the IM 30 . Unlike most of the ABC exporters, ABC importers in Gram-negative bacteria require periplasmic SBPs that provide speci city and high-a nity. In addition, it is widely accepted that the direction of substrate transport of ABC transporters can be predicted on the basis of both the sequence of the nucleotide-binding component (ATPase) 57,58 and the transmembrane-domain fold of the permease component 59 . The close orthologs in E. coli and Mycobacterium tuberculosis of the P. aeruginosa ATPase Ttg2A, MlaF (60% identity) and the Mce protein Mkl (40% identity), respectively, have sequence signatures typically found in prokaryotic ABC import cassettes 20,58 . Moreover, the remote homolog TGD3 from Arabidopsis thaliana is also a component of an ABC transport system (TGD) that imports phosphatidic acid to the chloroplasts through its outer and inner envelopes 60 . On the other hand, and although operon ttg2 resembles the classic organization of an ABC importer, there are evidences at the amino acid sequence level supporting evolution toward anterograde function. For instance, structural similarity searches with Dali (see Text S1) for the Ttg2B ortholog protein in A. baumannii (MlaE, PDB 6IC4 chains G and H) 30 , identi ed as best match a structure of the human ABCA1 (PDB 5XJY), a known ATP-binding cassette cholesterol/phospholipid exporter 61 . All things considered, we should not rule out the possibility of anterograde, in addition to retrograde, phospholipid tra cking by the Ttg2/Mla system. One possibility would be that of a countercurrent model 62 , in which different types of phospholipids would exchange between the two membranes obeying to a gradient (Fig. 7). A countercurrent model would explain how asymmetries in membrane lipid distribution might be achieved by the Ttg2/Mla system, but the direction in which each phospholipid is transported would need to be determined. In the anterograde transport proposed for E. coli 32 , MlaC is loaded by MlaD, suggesting that, for this to be e cient, MlaC should not bind free phospholipids. Taken back to the P. aeruginosa system, we have shown that Ttg2D Pae can load free cardiolipin in vitro, but have no data for the spontaneous binding of two diacyl phospholipids. In this system, the idea that the loading of two molecules may require a helper protein (the MlaD ortholog Ttg2C) seems plausible. This would facilitate a putative mechanism in which the Ttg2 system would contribute to the retrograde transport of single lipids (cardiolipin, maybe diacyl lipids also) as well as to the export, enabled by Ttg2C, of pairs of diacyl lipids to the OM (Fig. 7). The exact function of VacJ (MlaA equivalent) and its relation with the transport in either direction is still to be determined. While P. aeruginosa's vacJ gene is located outside the ttg2 operon, we have data demonstrating that strains lacking this gene share the same phenotype shown by ttg2 mutants. In E. coli, MlaA forms an active complex with the outer membrane proteins OmpC and OmpF 22,24,63 . However, in P. aeruginosa there are no clear orthologs to either of these porins, increasing the singular characteristics of this system in this species and suggesting potential mechanistic differences with the more studied E. coli transporter (Fig. 7).
We have provided additional evidence, based on NPN-uptake and antimicrobial susceptibility assays, that the Ttg2 system controls the permeability of the OM in P. aeruginosa regardless of genetic background. Cellular studies showed that deletion of Ttg2D speci cally increases the susceptibility to polymyxin, uoroquinolone, chloramphenicol and tetracycline antibiotics in the PAO1 reference strain and in three MDR clinical strains ( Fig. 6 and Table 1). This mutant phenotype was observed both in the presence and absence of speci c resistance mechanisms providing high-level resistance. For example, PAO1 is a relatively sensitive strain and LESB58 is a MDR strain, and both show diversity in their resistomes 64 .
Thus, for the strain and antibiotic panel considered, the increase in susceptibility upon ttg2 deletion seems to correlate with the antibiotic class rather than with the genetic background. This is in line with the physico-chemical properties of these antimicrobial compounds. Albeit positively charged, colistin is a signi cantly hydrophobic antibiotic that appears to gain access to the IM by permeating through the OM bilayer, while tetracyclines, chloramphenicol and quinolones use a lipid-mediated or a porin-mediated pathway depending on protonation state 65 . These antibiotic classes are classi ed within the same group of molecules according to their interactions with the cell permeability barriers 9 . The fact that other relatively hydrophobic antibiotics such as aminoglycosides are unaffected by the disruption of the Ttg2 system speaks in favor of the observed correlation between membrane phospholipid content and speci c susceptibility to certain antibiotics 52,53 . Another hypothesis that would explain the different impact of Ttg2 disruption on different antibiotic classes would be the possibility that phospholipids carried by the Ttg2 system, particularly cardiolipin, may interact with or stabilize certain e ux pumps in P. aeruginosa. A P. putida cardiolipin synthesis mutant was more sensitive to several antibiotics and to toluene, probably due to structural alterations in the RND e ux pumps 66 . Indeed, the protein composition of the OM can also have a strong impact on the sensitivity of bacteria to the different antibiotic classes 65 . It was probably the secondary effects of the removal of Ttg2D function that led the authors of another study, based on results on a Ttg2A mutant, to conclude that the function of the Ttg2 system in P. aeruginosa was associated with the export of antibiotics such as tetracycline out of the cell 33 . Although further investigations are still required, the activity of the Ttg2 system on membrane phospholipid homeostasis appears to be partly responsible for the lower basal susceptibility of P. aeruginosa to antimicrobial agents, particularly to polymyxins (see supplementary discussion).

Bacterial strains
All bacterial strains used in this study are provided in supplementary Table S6 and growth conditions in supplementary Text S1.
Expression, puri cation and preparation of Ttg2D Pae protein variants Recombinant Ttg2D from P. aeruginosa (Ttg2D Pae ) was obtained in the cytoplasm of Escherichia coli BL21(DE3) using the pET-based expression system and was puri ed to >99% purity. A P. aeruginosa PAO1 mutant lacking ttg2D was used for the homologous expression of a His-tagged variant of the protein in its natural environment and its subsequent puri cation. Periplasmic protein preparation from P. aeruginosa was done by sucrose-lysozyme method 67 . Both protein variants are tagged with a C-terminal 6-histidine tail for puri cation by a nity chromatography. Detailed methods for proteins expression and production are available in supplementary Text S1. Samples containing both homologously and heterologously produced proteins were desalted with the appropriate buffer on centricon micro concentrators for MS analyses.
Ttg2D Pae structure resolution Recombinant Ttg2D Pae obtained in E. coli was used to produce crystals for structure determination.
Methods for crystallization, data collection and structure re nement are available in supplementary Text S1. The data collection, processing, and re nement statistics are given in supplementary Table S1.
Atomic coordinates and structure factors have been deposited in the PDB with entry code 6HSY.

Delipidation of puri ed recombinant Ttg2D Pae
Recombinant protein produced in E. coli, diluted 1:1 with 1% TFA, was delipidated using an HPLC system and a C18 column (Phenomenex Jupiter 5U C18 300A) in 0.1% TFA. Protein was eluted with a gradient of acetonitrile, 0.1% TFA (monitored at 214 and 280 nm) and its delipidation was con rmed by native MS analyses (Fig. 4 inset). Delipidated protein was lyophilized, and typically resuspended in 100 mM NaCl, 10 mM Tris-HCl (pH 8.5), to counteract the acidity of TFA, before exchanging the buffer to the desired one.
Native mass spectrometry analysis and identi cation of bound phospholipids Native MS experiments were performed using a Synapt G1-HDMS mass spectrometer (Waters, Manchester, UK) at the Mass Spectrometry Core Facility of IRB Barcelona. Prior to the analysis, samples were desalted with 100 mM ammonium acetate on centricon micro concentrator. Samples were infused by automated chip-based nanoelectrospray using a Triversa Nanomate system (Advion BioSciences, Ithaca, NY, USA) as interface. After ion isolation, fragmentation was performed by CID (collision induced dissociation) in the transfer or trap region by applying increasing collision energies. See supplementary material (Text S1) for method details. Three technical replicates were performed for native MS experiments.
Analysis of protein Ttg2D Pae variants and their bound phospholipids was also done under denaturing conditions by electrospray ionization (ESI) MS in both the positive and negative ion mode (see Text S1).
Fragmentation analysis of the most abundant and representative glycerophospholipids released from Ttg2D Pae were done under denaturing MS conditions (non-native) in positive mode. Annotation of most abundant phospholipids present in each sample was done using Lipidomics Gateway (http://www.lipidmaps.org) based on the m/z values of MS spectra and according to Oursel et al. 44 and Gidden et al. 45 for E. coli phospholipids, and Groenewold et al. 47 for P. aeruginosa phospholipids. In addition, to determine the phospholipid composition from both E. coli BL21(DE3) and P. aeruginosa PAO1, and to identify the most detected phospholipid species by MS, lipidome analysis of intact cells was done according to Angelini et al. 68 (see Text S1).
Delipidated protein was mixed with cardiolipin at 1:9 molar ratio (26.25 μM:245 μM) and incubated at 37°C for 1 h at 500 rpm. The excess of cardiolipin were cleaned changing the buffer to 200 mM ammonium acetate using centrifugal lter units of 3K cut-off (Amicon Ultra, Millipore). Buffer was exchanged 3 times at 15°C at 13000g. The reaction was directly injected for MS analysis. Three technical replicates were measured. To check the behavior of cardiolipin and its fragmentation patterns in the different MS conditions applied, commercial CL(18:0) 4 has been used as a standard in all assays.
Generation of markerless ttg2 mutants in MDR P. aeruginosa strains and complementations Markerless P. aeruginosa mutants were constructed using a modi cation of the pGPI-SceI/pDAI-SceI system (Fig. S13) originally developed for bacteria of the genus Burkholderia and other MDR Gramnegative organisms 49,69 . The bacterial strains and plasmids of the pGPI-SceI/pDAI-SceI system were kindly donated by Uwe Mamat (Leibniz-Center for Medicine and Biosciences, Research Center Borstel, Borstel, Germany) with permission of Miguel A. Valvano (Center for Infection and Immunity, Queen's University, Belfast, UK). The pGPI-SceI-XCm plasmid 48 was rst modi ed to facilitate the generation of ttg2 mutants in MDR P. aeruginosa strains. Plasmid modi cations include replacement of the chloramphenicol resistance cassette by an erythromycin resistance cassette and deletion of a DNA region containing the Pc promoter found in P. aeruginosa class 1 integrons (Text S1 and supplementary Table  S6 for details). The sequence of the new suicide plasmid vector, pGPI-SceI-XErm, is available through GenBank under the accession number KY368390. For complementation in PAO1, full ttg2 operon or the codifying region of the ttg2D gene were cloned into the broad-host-range cloning vector pBBR1MCS-5 or a variant thereof containing the arabinose promoter, respectively (Table S6). For complementation experiments in MDR strains, the cloning vector pBBR1MCS-5 was rst modi ed to confer resistance to erythromycin (see details in Text S1). Sequence for the new cloning vector, pBBR1MCS-6 is available through GenBank under the accession number KY368389. Complemented strains were obtained by transforming mutant cells with the corresponding pBBR1MCS derivative plasmid. The expression of ttg2D in mutant and complemented strains was veri ed by reverse transcription-PCR (RT-PCR) and quantitative real-time RT-PCR analysis (Text S1 and Fig. S13).
Outer membrane permeabilization assay Fluorometric assessment of outer membrane permeabilization was done by the 1-Nphenylnaphthylamine (NPN) uptake assay as described by Loh et al. 70 with modi cations (Text S1).
Since P. aeruginosa PAO1 cells have proven to be poorly permeable to NPN 9 , either EDTA (0.2 mM) or colistin (10 μg ml -1 ) was added to cells to enhance uptake and uorescence.
Susceptibility to antibiotics and membrane-damaging agents Antimicrobial susceptibility to a range of antibiotics was tested by determination of the minimum inhibitory concentration (MIC) using the broth microdilution method or Etest (Biomerieux) strips, following the Clinical and Laboratory Standards Institute (CLSI) guidelines 71, 72 and manufacturer's instructions, respectively (see Text S1 for details). MIC differences higher than 2-fold were considered signi cant changes in antibiotic susceptibility. Low-level, basal resistance to a given antibiotic was de ned as that of an organism lacking acquired mechanisms of resistance to that antibiotic and displaying a MIC above the common range for the susceptible population 73 . Clinical susceptibility breakpoints against Pseudomonas sp. for selected antibiotics have been established by EUCAST 74 . Tolerance to organic solvents and SDS/EDTA was assessed using solvent overlaid-solid medium and MIC assays, respectively (Text S1).

Bio lm formation
Bio lm quanti cation in 96-well microtiter plate by the crystal violet assay was done as previously described 75 with modi cations (supplementary Text S1).

Bioinformatic analysis
Details are provided in the supplementary Text S1.